Larvicidal Activity of Bunium persicum Essential Oil and Extract against Malaria Vector, Anopheles stephensi.

Background
Malaria, a mosquito-transmitted disease, is still a major human health problem all over the world. Larviciding is a component of comprehensive control program to overcome the disease. Negative aspects of synthetic insecticides application, such as environmental safety concerns, have favored use of natural insecticides.


Methods
Larvicidal activity of essential oil, extracts and fractions of a wild grown and a cultivated type of Bunium persicum fruits against malaria vector Anopheles stephensi was assessed according to the method described by WHO.


Results
Bunium persicum showed remarkable potency against An. stephensi larvae. LC 50 values for essential oil, total extract, petroleum ether fraction and methanol fraction were 27.4284, 64.9933, 85.9933 and 255.7486ppm for wild type, and 21.3823, 63.2580, 62.7814 and 152.6357ppm for cultivated one.


Conclusion
The results of this study suggest B. persicum as a valuable source of natural insecticides against malaria vector Anopheles stephensi.


Introduction
Despite progresses made over the past decades to decline the mortality rate of malaria all over the world, it is still prevalent in some tropical countries and areas with about 200 million affected cases in 2013. Vector control interventions have had substantial contribution on the recent reduction in global malaria burden. Larviciding, with the aim of adult vector density reduction, as an auxiliary to core interventions, is helpful especially in urban regions, where breeding of vectors take places in permanent or semi-permanent aquat-ic habitats (1). The mosquito Anopheles stephensi is one of the six main vectors of human malaria in southern parts of Iran (2). Larvicidal potentials of some herbal extracts and essential oils on An. stephensi larvae have been investigated previously (3)(4)(5).
Bunium persicum is a perennial plant belonging to Apiaceae family, growing wild in Iran (6). The fruit of B. persicum is used as spice, antiseptic and carminative agent (7). Several studies have analyzed essential oil composition of the fruits and mostly reported γ-ter-pinene, cuminaldehyde and ρ-cymene as main components (8)(9)(10). Kaempferol, caffeic and pcoumaric acid have been isolated from polar fraction of the fruits as major antioxidant constituents (11) but according to our knowledge no other comprehensive study has been organized to identify other phytochemicals in the extract. Overexploitation and unscientific harvesting of B. persicum as well as climate changes, has threatened its existence in wild (12). Cultivation of endangered species could preserve their genetic resources (13). In recent years, B. persicum is cultivated in limited areas in Iran especially in Khorasan Razavi Province. As a part of our ongoing studies on larvicidal activity of plants extracts and essential oils against An. stephensi (4,5,(14)(15)(16)(17)(18)(19)(20), in the present study, we have studied larvicidal activity of the essential oil, extract and fractions from B. persicum fruits against late third instar larvae of An. stephensi. Moreover, we have compared the activities of a wild and a cultivated type.

Plant material
The fruit of wild B. persicum was purchased from Kerman, and cultivated type was supplied from agricultural research fields of Ferdowsi University of Mashhad (2013). The samples were authenticated at the Herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences, where voucher specimens were deposited (PMP-649 and PMP-689).

Essential oil preparation
100g powdered fruits of cultivated and wild B. persicum were subjected to hydrodistillation for 3 hours using Clevenger type apparatus. The obtained essential oils were dried over anhydrous sodium sulfate and kept in refrigerator until needed.

Extraction and fractionation
250g dried and powdered fruits from both samples were separately extracted with meth anol (5× 1.5L) to afford total methanol extracts. The solvent was removed under reduced pressure by rotary evaporator at 40 °C, and subsequently lyophilized by freeze dryer at -40 ºC for 24h (Lyotrap Ultra, LTE Scientific Ltd., Oldham, UK). Fractionation of total extracts was performed with sufficient volumes of petroleum ether, ethyl acetate and methanol. The fractions were then concentrated to dryness by rotary evaporation.

Larval mortality bioassay
Anopheles stephensi larvae (Bandar Abbas strain) were supplied by the Department of Medical Entomology, Tehran University of Medical Sciences. The mosquito colony was maintained under a constant insectarium condition at 27 ºC and 75-85% relative humidity with 12:12 light and dark photoperiod. Late third and early fourth instars larvae were used for experiments.
Larvicidal activity of total extracts, fractions and essential oils were evaluated according to the procedure recommended by WHO (21). The larvae were exposed to different concentrations of samples for 24 hours. Tests were carried out in four replicates. One ml of solvents (DMSO for essential oil and petroleum ether fraction, DMSO2: water 3 for total extract and ethanol for methanol fraction) were added separately into control bakers. Mortality was scored 24 hours post exposure.

Analysis method
The mortality percentages were calculated and corrected relative to the associated controls using Abbott's formula (22). The concentration-mortality data were subjected to Probit analysis (23) and lethal concentrations (LC50 and LC90) were determined with 95% confidence intervals from the regression lines.

Discussion
Many researchers have already studied larvicidal potentials of plant derived compounds, extracts and essential oils against various insects, with the aim of finding active phytochemicals to replace synthetic insecticides. High cost of various commercial insecticides beside their food and environmental safety concerns, toxicity problems and increasing resistance rates have made their utilization undesirable (25,26). Anti-larval activity of essential oil from a wild grown B. persicum against An. stephensi and Culex pipiens has been previously reported with LC50 values of 27.72 and 20.61ppm respectively (27). According to the results of our study, the essential oil, methanol total extract and petroleum ether fraction of both wild and cultivated samples had significant larvicidal activity against late third and early fourth instar larvae of An. stephensi. The larvicidal potential of γ-terpinene, cuminaldehyde and ρ-cymene, main constituents of both wild and cultivated type B. persicum fruits, against various insect larvae has been previously proved in several experiments. γ-Terpinene has shown potent larvicidal activity with LC50 value of 29.21 ppm against Anopheles anthropophagus (28) and 30.7 and 29.8ppm against Aedes aegypti and Aedes albopictus respectively (29). Zahran and Abdelgaleil (30) documented toxicity of cumin aldehyde on Culex pipiens larvae, which was more stronger than other tested monoterpenes in that experiment, with LC50 values of 38.9 and 21.4ppm for 24 and 48h exposures respectively. Anti-larval potential of ρ-cymene, the other main constituent, towards A. aegypti and Ae. albopticus has also been demonstrated (LC50= 19.2 and 46.7ppm) (29). Higher lethal effect of the petroleum ether fraction in comparison to the methanol fraction, suggests higher potency of non-polar components than polar phenolics towards An. stephensi larvae. LC50 value of 85.9933 and 62.7814ppm for petroleum ether fraction from wild and cultivated types makes it suitable choice for further studies to isolate the active principles. Anti-larval activity of efficient samples from cultivated type was comparable to those from wild grown, so it can be concluded that cultivation of B. persicum has not affected chemical constituents' biosynthesis or concentration, which are responsible for larvicidal activity of the fruit.

Conclusion
The extract and fractions from B. persicum fruits, ie, petroleum ether fraction and total extract, beside the essential oil, have shown significant larvicidal effects on An. stephensi, and can be a great candidate to develop an ecofriendly insecticide to combat malaria vector breeding. More precise investigation will require revealing phytochemical composition of extract. Since cultivated type showed comparable results as wild grown, cultivation of B. persicum, as a solution to preserve its wild resources, is highly recommended. There are several studies on larvicidal activities of different plants against malaria vectors in Iran (16,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). We recommend formulation of plant extract which have the lowest LC50 for field evaluation.